Actin depolymerizing factor (ADF) is a 19kDa protein which can sever actin filaments and sequester actin monomers. It is widely distributed among different tissues of the embryonic and adult chicken, and immunologically cross-reactive proteins of identical molecular mass occur in mammalian cells. ADF levels are high in embryonic tissue (as much as 0.6 mole ADF per mole of actin) but disappear from some adult tissues such as muscle, and decline 50% in others such as nerve. The ADF level does not decline during in vitro myogenesis, indicating one or more factors which regulate ADF synthesis are lacking. One of these factors is likely to be thyroid hormone which appears to regulate ADF expression in developing brain. ADF activity can also be controlled by posttranslational modifications. Several isoforms of ADF, both active and inactive, have been isolated from tissues and from cultured cells. Some cells have multiple active isoforms. These isoforms of ADF will be fully characterized with respect to activity, primary structure and posttranslational modifications, as well as to determine certain structure/function relationships within a single isoform of the molecule by site-directed mutagenesis of our cloned full length cDNA. This characterization will include a genomic analysis for the number and structure of the ADF gene(s), and the mechanisms by which the multiple mRNAs for ADF arise. The nature of the posttranslational regulatory system which controls ADF activity in vivo will be identified. The cellular function of ADF will be investigated by microinjection of ADF and activity- inhibiting antibodies. Specific antibody probes will be prepared which will allow us to analyze the temporal and spatial relationships between the inactivation of ADF and the assembly of actin in developing muscle. These studies will utilize chimeric myotubes formed from myocytes of mice carrying a dysgenic mutation and rescued by the insertion of a normal nucleus. The level at which ADF expression is regulated in myocytes and the effect of thyroid hormone on ADF expression will be determined. Successful completion of these studies will provide the means to determine the structure of actin in a filament and the role that the control of ADF expression and activity have in regulating actin assembly in many cell types.